Transcription factor mobility

许多重要基因的表达是周期性的，并且可能包含不同时间尺度的多个周期，例如小时尺度的昼夜节律或超昼夜周期，以及分钟尺度的快速和短的转录爆发。人们对这种循环的机制知之甚少，但对于药物的正确应用非常重要。在转录水平上，基因表达由 DNA 内调控元件（启动子和增强子）的可及性控制。对酵母和哺乳动物细胞的研究表明，染色质可及性的调节是通过转录因子 (TF) 和染色质重塑剂的相互作用发生的。某些转录因子与其目标 DNA 序列的结合是高度动态的，以秒为单位。此外，一些 TF 会经历分钟级的“缓慢”循环，其中包括交替的“ON”和“OFF”基因状态。目前尚不清楚这种 TF 循环与转录循环有何关系。我们对启动子上 TF 循环的分子机制及其与转录周期的相关性感兴趣。我们使用编码金属硫蛋白的酵母基因 CUP1 作为模型，该基因在重金属胁迫下表达。 CUP1 由铜结合 TF Ace1p 激活。我们之前的研究证明了 CUP1 上 Ace1p 循环的两种类型：快速（秒级）和慢速（分钟级）。快循环发生在慢循环内。结合的慢速循环可以通过常规荧光成像在单个细胞中量化，而快速循环则可以通过单分子追踪（SMT）来量化。此前，我们已经为 SMT 构建了定制仪器并优化了 SMT 数据解释。我们开发了在酵母细胞核中进行 SMT 的方法以及在特定启动子上进行 SMT 的方法。通过 SMT，我们测量了染色质重塑剂 RSC 和 TF Ace1p 与 CUP1 启动子内特定位点（金属响应元件，MRE）的结合参数。 CUP1 的转录可以通过 smFISH（荧光原位杂交）或荧光标记 mRNA 的实时成像来观察。最近，我们通过 smFISH 将 CUP1 启动子的转录活性与寻找结合位点和 TF 的特定停留时间的变化相关联。我们证明了转录因子的瞬时募集受到快速周期性染色质重塑事件的调节，以确保最佳的动态转录反应。我们当前的目标是通过活细胞转录分析来证实这些发现。初步观察表明，转录的慢周期由单个基因表达的快周期（爆发）组成。 smFISH 在固定细胞中无法进行此类观察，但提供了有关转录的新信息。这是第一个遗传模型，其中叠加的慢速和快速转录周期与叠加的转录因子慢速和快速循环相关。我们正在致力于通过实时 TS 对慢速和快速转录周期进行建模，并通过 smFISH 建模来证实观察结果。最终，这些研究将为分析转录机制各组成部分的体内相互作用奠定基础。 smFISH 数据通过茎环方法原位活转录定量得到证实。实时 TS 成像揭示了两种不同尺度的转录爆发——慢速和快速。值得注意的是，转录的慢周期与 TF 结合的慢周期相关。目前，我们正在表征转录的快速尖峰以及 TF 在这些短尖峰中的作用。我们正在开发的将转录因子生物物理学与转录模型相关联的技术可以应用于需要转录分子调控信息的细胞生物学的许多其他问题。

Expression of many important genes is cyclical, and may incorporate multiple cycles at the different time scale, such as circadian or ultradian cycles on the scale of hours, and fast and short bursts of transcription on the scale of minutes. The mechanisms of this cycling are poorly understood, but very important for the correct application of the drugs. At the transcription level, gene expression is controlled by the accessibility of the regulatory elements within DNA - promoters and enhancers. Research in yeast and mammalian cells indicates that modulation of chromatin accessibility occurs through interaction of transcription factors (TF) and chromatin remodelers. Binding of certain TF to their target DNA sequences is highly dynamic, on the scale of seconds. Also, some TF undergo the "slow" cycling on the scale of minutes, which consist of alternating "ON" and "OFF" gene states. It is unclear how this TF cycling is related to the transcriptional cycling. We are interested in molecular mechanisms of the TF cycling on promoters and its correlation with transcription cycles. We use as a model a yeast gene CUP1 encoding metallothionein expressed in response to heavy metal stress. CUP1 is activated by copper-bound TF Ace1p. Our previous studies demonstrated two types of Ace1p cycling at CUP1: fast - on the scale of seconds, and slow - on the scale of minutes. The fast cycling occurs within the slow cycle. The slow cycle of binding may be quantified in individual cells by the regular fluorescence imaging, and the fast cycling - by Single Molecule Tracking (SMT). Previously, we have built a custom instrument for SMT and optimized SMT data interpretation. We have developed methods for SMT in yeast cell nuclei and methods of performing SMT on specific promoters. By SMT, we have measured binding parameters of a chromatin remodeler RSC and TF Ace1p to specific sites (Metal Response Elements, MRE) within CUP1 promoter. Transcription of CUP1 may be observed either by smFISH (Fluorescence In Situ Hybridization) or by live imaging of the fluorescently tagged mRNA. Recently, we correlated the transcriptional activity of CUP1 promoter by smFISH with changes in the search for the binding sites and the specific residence time of TF. We demonstrated that the transient recruitment of TFs is regulated by fast cyclical chromatin remodeling events to ensure the best dynamic transcriptional response. Our current goal is to corroborate these findings with analysis of the transcription in live cells. Preliminary observations indicate that the slow cycle of transcription consists of the fast cycles (bursts) of expression on individual genes. This kind of observations cannot be made by smFISH in fixed cells and provides new information about transcription. This is the first genetic model where the superimposed slow and fast cycles of transcription correlate with superimposed slow and fast cycling of the transcriptional factor. We are working on modeling of the slow and fast transcription cycles by live TS and corroborating the observations by smFISH modeling. Ultimately, these studies will lay the groundwork for the analysis of in vivo interactions of the components of the transcriptional machinery. The smFISH data are corroborated by the live transcription quantification in situ by a stem-loop approach. Live TS imaging revealed transcription bursting on two different scales - slow and fast. Notably, slow cycle of transcription correlates with slow cycle of TF binding. Currently, we are characterizing fast spikes of transcription and the role of TF in those short spikes. The technique of correlation of the TF biophysics with transcription modeling that we are in process of developing may be applied to a number of other problems of cellular biology where the information for molecular regulation of transcription is desired.